Vitamin B 12 is an essential nutrient for humans that is probably synthesized only by some bacteria and archaea. The need and ability to synthesize B 12 is unevenly distributed in nature and the physiological significance of B 12 in the organisms that use it is uncertain. (It's not completely clear even for humans.) Some animals and apparently all plants and fungi do not need or use B12, others require it in their diet. We discovered that the all isolates of Salmonella (and no isolates of their near relative, E. coli) make B 12 (when growing anaerobically) and dedicate 1% of their genome to the synthesis and transport of this huge cofactor. This project is aimed at better understanding the synthesis of this vitamin and why its synthesis has been maintained by all the Salmonellae. Work includes experiments to refine our understanding of the synthetic pathway. Work on the uses of B 12 should provide insights into the anaerobic lifestyle of Salmonella and should suggest roles that B 12 may play in other organisms. The regulatory behavior of the B 12 synthetic genes (cob) suggests that the main use of B12 is in the anaerobic breakdown of two carbon sources, ethanolamine and propanediol. The project includes work on the synthetic genes for B 12 and two operons that encode the utilization pathways of ethanolamine (eut) and propanediol (pdu). Both of these catabolic operons encode multiple attendant proteins that appear to adenosylate of B12 and protect the relevant enzyme from inhibition by other B12 forms. There are indications that each pathway may involve a huge complex of proteins that forms an organelle designed to protect the enzymes from oxygen or to sequester Ado-Bl2. The mechanism by which the cob genes are repressed may involve recognition of B 12 by an RNA molecule and therefore may provide regulatory novelties that hearken back to the primitive origins of Bl2 in the "RNA world". The evolution of the Bl2 pathway has involved horizontal transfer of the synthetic genes into Salmonella from another bacterium. This acquisition may have been involved in the divergence of Salmonella from E coli and is correlated with acquisition of pathogenic lifestyle. The relevance of this project to human health lies both in providing insights into the synthesis of an essential human nutrient and in providing background information that may be important to understanding Salmonella's interaction with animal hosts. (Salmonella cob mutants are more pathogenic in mice than wild type strains. Salmonella eut mutations are impaired for ability to survive in a host macrophage.)